ABSTRACT
Over the last decade, five different proteomic technology platforms have been commonly used for protein separation: two-dimensional gel electrophoresis (2DE), surface-enhanced laser desorption/ ionization (SELDI), LC-MS, CE coupled to MS (CE-MS), and protein arrays. In 2DE, proteins are separated according to their isoelectric point and molecular weight. Protein identification is routinely performed on proteolytic in-gel digests, followed by peptide extraction from the gel and MS analysis [14]. To improve gel-to-gel protein spot matching accuracy and to increase the reliability of protein quantification, two-dimensional difference gel electrophoresis (2D-DIGE) was developed. The method uses two samples differentially labeled with fluorescent dyes (e.g., Cy3 and Cy5). Subsequently, the two samples are separated simultaneously within the same gel. In addition to satisfactory comparison of two samples [15], the comparison of several different experiments become possible using another labeled sample as a calibrator. Methodologically, 2DE is rather time consuming, due to a certain extent to the lack of automation, and comparability between laboratories has been difficult to achieve [16]. However, 2DE still appears to be the method of choice for comparative analysis of abundant proteins. SELDI was devised to overcome some of the limitations of 2DEMS and has been applied in a number of clinical investigations of the proteome. Numerous matrices are used, such as hydrophilic materials, reverse-phase materials, and affinity reagents. Low reproducibility of binding, the inability to detect low abundance peptides and proteins, and the inability to directly identify discriminatory peaks are major issues to be overcome. With the progress in high-resolution mass spectrometry and improvements in sample preparation and pre-fractionation strategies, efforts are on the way to enhance reproducibility [17, 18]. LC-MS provides high-resolution separation with a large capacity for analytes that can be loaded onto an LC column. Approaches like multidimensional protein identification technology [19] and 2D liquid phase fractionation [20] provide a great amount of data. However, LC-MS tends to be time consuming, which limits its application in routine clinical analysis. LC is sensitive towards interfering
compounds and the precipitation of analytes in the column [2]. In the last decade, microscale chromatographic separation techniques, such as L-LC-MS (20), have been designed to resolve these limita-tions-they permit easy column preparation, high permeability, low backpressure, fast analyte mass transfer, and versatile surface chem-istry [21]. CE-MS uses free-flow separation of analytes in buffer-filled capillaries [22]. Therefore, it is relatively robust and compatible with most volatile buffers without continuous adjustment of the ionization voltage for optimal ESI, mainly due to the absence of buffer gradients, as is the case in LC-coupled MS analysis. Therefore, CE-MS certainly appears to be an excellent choice for fast high-resolution analysis of complex biological samples. In contrast to the platforms already discussed above, detection of specific proteins with protein microarrays is a non-MS-based targeted proteomics approach. The technique imprints specific antibodies or antigens on a plate to facilitate immune detection of multiple proteins. A single sample is hybridized to the array followed by the detection of the captured antigens or antibodies [23]. However, the requirement for antibodies with accurate sequence information and with high specificity for target peptides limits usage in clinical analysis. 6.4 MS Techniques
A detailed overview of the different ionization processes and the recent advances in mass spectrometry is far beyond the scope of this review and will be introduced in another chapter. Briefly, ionization of polypeptides can technically be performed by electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI). ESI generates charged droplets in a high-voltage field. In this field, the solvent in the droplet evaporates, generating multiply charged analyte ions, which make it possible to analyze highmolecular-weight proteins. ESI pre-MS separation can be coupled online with a physical connection to an MS. This approach may be less stable compared with MALDI due to possible electrospray collapse, but it is less susceptible to the signal suppression phenomenon, in which certain analytes are preferentially ionized, and hence easily detected, but other analytes may become masked from detection [24].
